Recent discoveries of the roles of RNA-based silencing during virus-host interactions of multicellular eukaryotes have revealed fundamental mechanisms that govern virus susceptibility and defense. Silencing mechanisms operate to condition local and systemic antiviral states in response to infection, but also to modulate cellular or viral functions that enable virus replication, invasiveness and latency. Understanding these mechanisms will illuminate basic events that occur at the interface between virus and host, and will reveal natural antiviral defense mechanisms that may be exploited for directed therapies or preventative measures. The genetic, genomic and technical aspects of the Arabidopsis model have proven exceptionally useful in revealing RNA silencing functions that limit virus infection at the cell-autonomous and cell-nonautonomous levels, as well as silencing functions that govern stress responses, development, and repressive chromatin. During the current project period, Arabidopsis was used to identify the roles of cellular and viral factors, such as virus-encoded suppressors of RNA silencing, during antiviral defense and counter defensive processes. This research, and work from several other groups, led to conceptualization of a three-phase model for antiviral silencing in Arabidopsis. This model describes events occurring during the initial targeting phase, the siRNA amplification phase and the systemic silencing phase. The three Aims of the proposed project focus on virus-host interactions that occur during each phase. First, new high-throughput sequencing technology will be used to analyze the genetic requirements for initial targeting of viral genomes by DICER-LIKE (DCL) factors during early stages of infection, and to test the hypothesis that initial targeting yields primarily (+)-sense siRNA that seed the subsequent amplification step. Second, the genetic requirements and detailed accumulation patterns of siRNA formed during the RNA- DEPENDENT RNA POLYMERASE6-dependent amplification phase will be determined, and the small RNA populations that interact with a viral RNA silencing suppressor will be identified. And third, the hypothesis that viral silencing suppressors function in vascular cells to inhibit cell-nonautonomous, DCL4-dependent systemic signals that limit virus invasiveness will be tested.